title: Gephyrin plays a key role in BDNF-dependent regulation of amygdala surface GABAARs.
abstract: Brain-derived neurotrophic factor (BDNF) is critically involved in synaptic plasticity and neurotransmission. Our lab has previously found that BDNF activation of neurotrophic tyrosine kinase, receptor, type 2 (TrkB) is required for fear memory formation and that GABAA receptor (GABAAR) subunits and the GABAA clustering protein gephyrin are dynamically regulated during fear memory consolidation. We hypothesize that TrkB-dependent internalization of GABAARs may partially underlie a transient period of amygdala hyperactivation during fear memory consolidation. We have previously reported that BDNF modulates GABAAR α1 subunit sequestration in cultured hippocampal and amygdala neurons by differential phosphorylation pathways. At present, no studies have investigated the regulation of gephyrin and GABAAR α1 subunits following BDNF activation in the amygdala. In this study, we confirm the association of GABAAR α1 and γ2 subunits with gephyrin on mouse amygdala neurons by coimmunoprecipitation and immunocytochemistry. We then demonstrate that rapid BDNF treatment, as well as suppression of gephyrin protein levels on amygdala neurons, induced sequestration of surface α1 subunits. Further, we find that rapid exposure of BDNF to primary amygdala cultures produced decreases in gephyrin levels, whereas longer exposure resulted in an eventual increase. While total α1 subunit levels remained unchanged, gephyrin was downregulated in whole cell homogenates, but enhanced in complexes with GABAARs. Our data with anisomycin suggest that BDNF may rapidly induce gephyrin protein degradation, with subsequent gephyrin synthesis occurring. Together, these findings suggest that gephyrin may be a key factor in BDNF-dependent GABAAR regulation in the amygdala. This work may inform future studies aimed at elucidating the pathways connecting BDNF, GABAA systems, gephyrin, and their role in underlying amygdala-dependent learning.
authors.affiliation: Department of Psychiatry and Behavioral Sciences, Emory University School of Medicine, Atlanta, GA, USA; Howard Hughes Medical Institute, Chevy Chase, MD, USA.
authors.forename: L
authors.initials: L
authors.lastname: Mou
authors.forename: B G
authors.initials: BG
authors.lastname: Dias
authors.forename: H
authors.initials: H
authors.lastname: Gosnell
authors.forename: K J
authors.initials: KJ
authors.lastname: Ressler
mesh_terms: D000679:Amygdala / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D019208:Brain-Derived Neurotrophic Factor / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000502:physiology; D006801:Humans; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D009474:Neurons / Q000378:metabolism; D011963:Receptors, GABA-A / Q000378:metabolism*; D014162:Transfection
chemical_list: D019208:Brain-Derived Neurotrophic Factor; D002352:Carrier Proteins; D008565:Membrane Proteins; D011963:Receptors, GABA-A; C073441:gephyrin
keywords: BDNF; GABA; GABA(A) receptors; GABA(A)Rs; GFP; ICC; IP; PBS; SDS–PAGE; TrkB; amygdala; brain-derived neurotrophic factor; consolidation; fear; gephyrin; green fluorescent protein; immunocytochemistry; immunoprecipitation; memory; neurotrophic tyrosine kinase, receptor, type 2; phosphate-buffered saline; sodium dodecyl sulfate polyacrylamide gel electrophoresis